Build-a-GeneSession 5September 3, 2016

PCR purification

1.  Add 75 ul of water to one of your PCR products and mix by pipetting.

2.  Add 500 ul buffer PB to the tube and pipet up and down several times to mix.

3.  Obtain one spin column/collection tube and label with your initials. Add the liquid from step 2 to the top of the column.

4.  Spin in centrifuge for 1 min (be sure the centrifuge is balanced with another tube on the opposite side).

5.  Remove the spin column and pour out the liquid from the bottom collection tube. Put the spin column back in the collection tube.

6.  Add 750 ul buffer PE to the top of each column.

7.  Spin in centrifuge for 1 min (be sure the centrifuge is balanced with another tube on the opposite side).

8.  Remove the spin column and pour out the liquid from the bottom collection tubes.

9.  Repeat steps 6 and 7.

10.  Obtain a new 1.7 ml microcentifuge tube and label with your initials.

11.  Discard the collection tube and transfer the column to the new labeled microcentrifuge tube.

12.  Add 50 ul elution buffer to the column. The microcentrifuge tube cap will not close over the spin column, just leave the tube open.

13.  Wait 1 min and then spin the tube and column in centrifuge for 1 min.

14.  Throw out column. The liquid in the bottom of the microcentrifuge tube is your purified DNA. This purified DNA is now ready to be sent for sequencing.

Genomic DNA Preparation from Yeast using Wizard Genomic DNA Kit

1. Transfer 1 ml of a yeast culture to a microcentrifuge tube.

2. Centrifuge at full speed for 2 minutes to pellet the cells. Remove the supernatant (liquid).

3. Resuspend cells in 300 µl of Zymolyase solution and gently pipet up and down to mix until no

clumps remain.

4. Incubate the sample at 37°C for 60 minutes to digest the cell wall. Cool to room temperature.

5. Centrifuge the sample at full speed for 2 minutes and then remove the supernatant (liquid).

6. Add 300µl of Nuclei Lysis Solution to the cell pellet and gently pipet to mix.

7. Add 100µl of Protein Precipitation Solution and vortex vigorously at high speed for 20 seconds. Let the sample sit on ice for 5 minutes.

8. Centrifuge at full speed for 3 minutes to pellet the proteins and cellular debris.

9. Obtain a new microcentrifuge tube and transfer the supernatant containing the DNA into the clean tube. Note: Some supernatant may remain in the original tube. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.

11. Add 300µl of room temperature isopropanol to each sample. Gently mix by mixing the tube end-over-end several times. You may see thread-like strands of DNA form a visible mass as they solidify and come out of solution. Incubate at room temperature for 5 minutes.

12. Centrifuge at full speed for 2 minutes. This will cause the DNA and RNA to pellet at the bottom of the tube.

13. Carefully remove the supernatant with a P1000 pipet and discard the liquid. Add 300µl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.

14. Centrifuge at full speed for 2 minutes. Carefully remove the supernatant with a P1000 pipet.

15. Centrifuge at full speed briefly. Remove all remaining supernatant using a pipette. Leave the tube open on the lab bench for 10–15 minutes to allow all remaining ethanol evaporate.

16. Add 25µl of DNA Rehydration Solution. Add 1.5µl of RNase Solution to each sample and mix well. Centrifuge briefly in a microcentrifuge for 5 seconds to collect the liquid and incubate at 37°C for 15 minutes. The RNase enzyme will degrade the RNA during this time.

17. Store the DNA at 4°C or -20°C long term.